Cancer is a major cause of death in the world. Because traditional cancer therapy targets all rapidly dividing cells, these therapies can have devastating side effects because they affect non-cancerous cells such as cells of the gastrointestinal tract, immune system, and hair follicles. Therefore, new methods of treatment are needed that are able to more specifically target cancer cells and, as such, avoid the side effects typical of cancer therapy.
Drosophila toll proteins control dorsal-ventral patterning and are thought to represent an ancient host defense mechanism. In humans, TLRs are believed to be an important component of innate immunity. Human and Drosophila Toll protein sequences show homology over the entire length of the protein chains. The family of human Toll-like receptors is comprised of ten highly conserved receptor proteins, TLR1-TLR10. Like Drosophila toll, human TLRs are type I transmembrane proteins with an extracellular domain consisting of a leucine-rich repeat (LRR) domain that recognizes pathogen-associated molecular patterns (PAMPs), and a cytoplasmic domain that is homologous to the cytoplasmic domain of the human interleukin-1 (IL-1) receptor. Similar to the signaling pathways for both Drosophila toll and the IL-1 receptor, human Toll-like receptors signal through the NF-κB pathway.
Although the different mammalian TLRs share many characteristics and signal transduction mechanisms, their biological functions are very different. This is due in part to the fact that four different adaptor molecules (MyD88, TIRAP, TRIF and TRAF) are associated in various combinations with the TLRs and mediate different signaling pathways. In addition, different ligands for one TLR may preferentially activate different signal transduction pathways. Furthermore, the TLRs are differentially expressed in various hematopoietic and non-hematopoietic cells. Accordingly, the response to a TLR ligand depends not only on the signal pathway activated by the TLR, but also on the nature of the cells in which the individual TLR is expressed.
Although ligands for some TLRs remain to be identified, a number of TLR specific ligands have been reported. For example, TLR3 ligands (agonists) include double stranded RNA such as Poly IC and Poly AU. Polyinosinic-polycytidylic acid (Poly IC) is a high molecular weight synthetic double stranded RNA that is heterogeneous in size. Polyadenylic-polyuridylic acid (Poly AU) is a double stranded complex of synthetic polyribonucleotides. Both Poly IC and Poly AU have been used in several clinical trials as adjuvant therapy in different types of cancer, such as cancer of the breast, bladder, kidney and stomach.
Stimulation of TLR3 by double stranded RNA or other agonists in, e.g., dendritic cells or B lymphocytes leads to the production of cytokines such as IFN, the activation of the innate immune system (NK cells), the enhancement of CD8+ T cells, and antigen cross-priming by dendritic cells.
Accordingly, TLR3 plays an important role in the defense against viral infection, and agonists have been used as adjuvants for cancer therapy in the past (see, e.g., Lacour et al. (1980) Lancet 2: 161-164; Khan et al., (1995) Eur. J. Surg. Oncol. 21:224-227).
While most studies of human TLRs, e.g., TLR3, have focused on their role in immune cells, it is now clear that they are also widely expressed in non-immune cells, including transformed cells such as breast cancer, cervical cancer, hepatomas, and melanomas, among others. Indeed, there is evidence that the efficacy of TLR3 agonists such as poly-AU or poly-IC in cancer therapy is not necessarily based on immune cell activation but on the induction of apoptosis in TLR3-expressing tumor cells (see, e.g., Salaun et al. Clin Cancer Res (2007); 4565 13(15) Aug. 1, 2007; Salaun et al., 2006, The Journal of Immunology, 176: 4894-4901; the entire disclosures of which are herein incorporated by reference). Because such TLR3 agonist therapy depends on the expression of TLR3 in the tumor cells, therefore, it is of obvious utility to be able to reliably and easily detect TLR3 levels in tumor cells.
A simple and practical way of detecting the expression of specific proteins in vivo is by immunostaining of paraffin-embedded tissue sections. Using this method, thin sections of tissue (e.g., cancer tissue obtained by biopsy) are obtained, fixed in e.g., formalin, embedded in paraffin, and then cut into very thin sections and mounted on slides. Following deparaffination, the slides are amenable to, e.g., immunohistochemical methods to detect the expression of specific proteins. This method is particularly useful because it gives rise to stable preparations in which the specific cellular and intracellular localization of specific proteins can be assessed.
Unfortunately, however, it is not always possible to find antibodies, particularly monoclonal antibodies, that work effectively and specifically in paraffin-embedded sections. Indeed, it is often much more difficult to obtain useful antibodies for immunohistochemistry on paraffin-embedded slides than it is for antibodies for use in other detection methods such as immunoblotting. The present invention addresses these and other needs.